Treatment of synucleinopathies

ABSTRACT

This invention relates generally to treating synucleinopathies in subjects that are not clinically diagnosed with a lysosomal storage disease, as well as associated methods of making medicaments and screening methods.

This application claims the benefit of International Application NumberPCT/US2008/064017, filed on May 16, 2008, U.S. Provisional ApplicationNo. 60/930,462, filed on May 16, 2007, and U.S. Provisional ApplicationNo. 60/929,554, filed on Jul. 3, 2007, all of which are incorporatedherein by reference in their entirety.

TECHNICAL FIELD

This invention relates generally to treating synucleinopathies that arenot lysosomal storage diseases in subjects, as well as associatedscreening methods.

BACKGROUND

Genetic, neuropathological, and biochemical evidence has implicated anincreased steady-state abundance as well as aberrant processing ofα-synuclein (αS) in the development of several neurodegenerativedisorders including Parkinson disease (PD), dementia with Lewy bodies(DLB), and others (Dawson et al., (2003) Science 302, 819-22; Vila etal., (2004) Nat. Med., 10 Suppl: S58-62).

Genetic evidence demonstrates that point mutations in theα-synuclein-encoding gene are linked to a severe, dominantly-inheritedform of PD with early onset (Krueger et al., (1997) Nat. Genet., 18,106-108; Zarranz et al., (2004) Ann. Neurol., 55(2):164-73;Polymeropoulos et al., (1997) Science, 276:2045-7), implying a“toxic-gain-of-function” pathogenesis. These mutations cause thefollowing amino acid changes: alanine 30→proline (A30P), glutamine46→lysine (E46K), and alanine 53→threonine (A53T). Furthermore,duplication and triplication of the α-synuclein encoding synuclein,alpha (non A4 component of amyloid precursor) gene (SNCA) have beenlinked to familial parkinsonism with a combined PD/DLB phenotype, whichdemonstrates that increased expression rates of even the wild-type (wt)gene can cause disease (Chartier-Harlin et al., (2004) Lancet, 364,1167-9; Singleton et al., (2003) Science, 302, 841). Intriguingly,certain polymorphisms within the promoter region of the SNCA gene havealso been linked to increased risk for sporadic, late-onset PD (Pals etal., (2004) Ann. Neurol., 56, 591-5; Maraganore et al., (2006) JAMA,296, 661-70).

Neuropathological evidence indicates that the intra-neuronal inclusionstermed Lewy bodies and Lewy neurites, which represent one of thepathological hallmarks of PD and DLB seen at autopsy, contain highlevels of aggregated α-synuclein protein (Spillantini et al., (1998)Proc. Natl. Acad. Sci., U.S.A., 95, 6469-73; Baba et al., (1998) Am. J.Pathol., 152, 879-884). These aggregates are generally viewed as theresult of cellular mis-handling of α-synuclein protein (possibly relatedto post-translational events, such as hyper-phosphorylation (Anderson etal., (2006), J. Biol. Chem., 281, 29739-29752) and intracellularaccumulation as both soluble toxic oligomers and insoluble fibrils(Sharon et al., (2001), P.N.A.S., 98, 9110-9115).

In addition, biochemical evidence suggests that overexpression ofα-synuclein in cellular or animal systems may cause cellular stressand/or eventual death through a variety of mechanisms, including—amongothers—excess dopamine concentration and reactive oxygen speciesgeneration (Tabner et al., (2002), Free Radic. Biol. Med.,32(11):1076-83; Fahn et al., (1992), Ann. Neurol., 32, 804-12) as wellas mitochondrial dysfunction (Lee (2003), Antioxid. Redox Signal,5:337-48; Hashimoto et al., (2003), Neuromolecular Med., 4(1-2):21-36).Published PCT patent application WO 07084737 discloses treatinglysosomal storage disorders having central nervous system implicationswith lysosomal enzymes.

SUMMARY

The invention is based, at least in part, on the discovery that certainagents, including acid-beta-glucocerebrosidase (GBA) polypeptides andselect members of the cathepsin family of proteases (e.g., cathepsin D)can reduce the intracellular levels of alpha-synuclein (αS) withinelements of the central and/or peripheral nervous system. As a result,the invention includes, inter alia, new methods of treatingsynucleinopathies, e.g., primary synucleinopathies, in subjects withouta known classical lysosomal storage disorder, e.g., by administering anon-protease-type lysosomal enzyme polypeptide, e.g., alipid-metabolizing enzyme, such as a GBA polypeptide, or a nucleic acidmolecule that encodes a GBA polypeptide, or agents that activate GBAactivity, or a protease-type lysosomal enzyme that hasalpha-synuclein-lowering activity (“synucleinase” activity).

In general, protease-type lysosomal enzymes fall into the categories ofaspartyl proteases (such as a cathepsin D or cathepsin E), and cysteinylproteases (e.g., cathepsin F and cathepsin L). Therefore, the inventionalso includes, inter alia, new methods of treating synucleinopathieswith protease-type lysosomal enzymes as well as procathepsin D, E, F,and L polypeptides, or nucleic acid molecules that encode cathepsin D,E, F, or L, or those that encode their pro- and pre-pro-proteinpolypeptide forms.

In addition, non-protease enzymes, e.g., GBA polypeptides, or proteaseenzymes, such as cathepsin D polypeptides, can be co-administered withagents that enhance or induce autophagy, such as rapamycin or rapamycinanalogs.

Moreover, given the pivotal roles that prosaposin (PS) and itsderivatives, saposin A (SA), saposin B (SB), saposin C (SC), and saposinD (SD), play as co-factors in the activity of GBA in vivo, othertherapeutic methods include administering GBA polypeptides together withGBA-activating polypeptides, such as PS polypeptides and/or SCpolypeptides; or administering PS polypeptides and/or SC polypeptidesalone (to activate or enhance endogenous GBA) to facilitate a reductionin α-synuclein steady-state protein levels in vivo.

In general, the invention features methods of treating subjects, e.g.,humans or animals, such as domesticated animals, e.g., dogs, cats,horse, goats, cows, and pigs, with a synucleinopathy, e.g., a primary orsecondary synucleinopathy, but not a clinically diagnosed, or not aclinically diagnosable, lysosomal storage disease. These methods includeadministering to a subject any one or more of: a lysosomal enzymepolypeptide (e.g., a non-protease-type polypeptide such as GBA or aprotease-type enzyme polypeptide such as cathepsin D), a polynucleotideencoding one or more lysosomal enzyme polypeptides, a lysosomal enzymeactivating agent, and a polynucleotide encoding a lysosomal enzymeactivating agent, in an amount effective to reduce a level ofα-synuclein in the subject's central or peripheral nervous system, orboth, or in the subject's lysosomal compartment.

The synucleinopathy can be any one or more of: Parkinson's disease (PD);sporadic or heritable dementia with Lewy bodies (DLB); pure autonomicfailure (PAF) with synuclein deposition; multiple system atrophy (MSA);hereditary neurodegeneration with brain iron accumulation; andincidental Lewy body disease of advanced age. In other embodiments, thesynucleinopathy can be any one or more of: Alzheimer's disease of theLewy body variant; Down's syndrome; progressive supranuclear palsy;essential tremor with Lewy bodies; familial parkinsonism with or withoutdementia; tau gene and progranulin gene-linked dementia with or withoutparkinsonism; Creutzfeldt Jakob disease; bovine spongiformencephalopathy; secondary Parkinson disease; parkinsonism resulting fromneurotoxin exposure; drug-induced parkinsonism with α-synucleindeposition; sporadic or heritable spinocerebellar ataxia; amyotrophiclateral sclerosis (ALS); and idiopathic rapid eye movement sleepbehavior disorder.

In these methods, the protease-type lysosomal enzyme can be an aspartylprotease polypeptide, such as a cathepsin D polypeptide, a procathepsinD polypeptide, a cathepsin E polypeptide, and a procathepsin Epolypeptide, or cysteinyl protease polypeptide, such as cathepsin Fpolypeptide, a procathepsin F polypeptide, a cathepsin L polypeptide,and a procathepsin L polypeptide.

In certain embodiments, the lysosomal enzyme activating agent is orincludes a GBA polypeptide activating agent, such as isofagomine (IFG),or activating polypeptide, such as any one or more of a prosaposinpolypeptide, a saposin A polypeptide, a saposin B polypeptide, a saposinC polypeptide, and a saposin D polypeptide. Of course, a polynucleotideencoding any one or more of a prosaposin polypeptide, a saposin Apolypeptide, a saposin B polypeptide, a saposin C polypeptide, and asaposin D polypeptide can also be used.

In another aspect, the invention features methods of treatingsynucleinopathies, as described herein, and by further administering oneor more agents that enhance autophagy of the α-synuclein. For example,the agent can be or include an mTOR inhibitor, rapamycin, a rapamycinanalog, everolimus, cyclosporine, FK506, hsc70,N-octyl-4-epi-β-valienamine, or glycerol.

In the methods described herein, the agents can be a small molecule, alarge molecule, a peptide, an antibody, a nucleic acid, or abiologically active fragment thereof.

In another aspect, the invention includes the use of any one or more ofa lysosomal enzyme polypeptide, a polynucleotide encoding one or morelysosomal enzyme polypeptides, a lysosomal enzyme activating agent, anda polynucleotide encoding a lysosomal enzyme activating agent, asdescribed herein, in methods of preparing medicaments for the treatmentof a synucleinopathy, using well known methods of manufacture.

By “polypeptide” is meant any chain of amino acids, regardless of lengthor post-translational modification (e.g., glycosylation orphosphorylation), and thus includes proteins, polypeptides, andpeptides.

By a “substantially pure polypeptide” is meant a polypeptide that hasbeen separated from components which accompany it in vivo. A polypeptideis substantially pure when it is at least 60%, by weight, free from theproteins and naturally-occurring organic molecules with which it isnaturally associated. Preferably, the preparation is at least 75%, morepreferably at least 90%, and most preferably at least 99%, by weight, ofthe desired polypeptide.

A “GBA polypeptide” is any GBA protein or polypeptide that has at least50 percent of the biological activity of the corresponding wild-type GBAin reducing a level of αS in a dopaminergic cell model as describedherein.

A “cathepsin polypeptide,” such as a cathepsin D polypeptide is anycathepsin protein or polypeptide that has at least 50 percent of thebiological activity of the corresponding wild-type cathepsin in reducinga level of αS in a dopaminergic cell model as described herein.

An “alpha-synuclein” protein or polypeptide (αS or αS protein), as usedherein, includes a single, monomeric protein or polypeptide, as well assuch αS proteins and polypeptides in the form of oligomers, e.g., in theform of dimers or trimers, or in the form of lipid-associated complexes,or lipid-free forms, or in the form of aggregates, and any of theseforms can be soluble or insoluble. The terms also include the αSproteins found in complexes with other molecules.

In another aspect, the invention features methods for identifyingcandidate compounds for treating a synucleinopathy, including (a)obtaining a model system, e.g., a cellular system, such as adopaminergic cell model, e.g., as described herein, facilitating thequantification of α-synuclein complexes; (b) contacting the model systemwith a test compound for incubation; and (c) comparing a level ofα-synuclein in the presence and in the absence of the test compound;wherein a decrease in the level of α-synuclein complexes in the presenceof the test compound indicates the test compound is a candidate compoundfor treating a synucleinopathy. In some embodiments, the precisequantification of α-synuclein protein is accomplished by the employmentof a sandwich-type, specific and sensitive ELISA, e.g., as describedherein. The α-synuclein model system can be, e.g., a protein-expressingcell or an animal model.

The invention also features methods of treating synucleinopathies,wherein the number or concentration of glucosylceramide andglucosylceramide-containing glycosphingolipids is reduced within neuraland non-neural cells by targeting glucosylceramide andglucosylceramide-containing glycosphingolipids with proteins, peptidesequences, enzymes, antibodies, natural lipids, semi-synthetic lipids,and synthetic lipids as well as derivatives thereof. For example, thenumber or concentration of glucosylceramide andglucosylceramide-containing glycosphingolipids may be reduced withinneural and non-neural cells by enzymatic or non-enzymatic hydrolysis ofglucosylceramide and glucosylceramide-containing glycosphingolipids.Such methods can be catalyzed by GBA in either a wild-type form or in amutant form that is binding-competent, but catalytically inactive. Inthese methods, prosaposin and/or its derivatives, such as saposin C, asdescribed herein can also be administered. In some embodiments, thedesired protein or polypeptide, such as GBA, is obtained expression of apolynucleotide encoding the enzyme or a derivative thereof.

In these methods, the agents, such as prosaposin, saposin A, saposin B,saposin C, saposin D, peptides derived thereof, small- or largemolecules, antibodies, fragments of antibodies or small or largepolynucleotides that improve the natural biological function of GBA,e.g., GBA activating agents, are delivered to the central and/orperipheral nervous system in an amount effective to decrease a level ofαS protein.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a representation of a Western Blot, demonstrating the timedependent formation of a stable 19-20 kDa complex (αS/G) between humanbrain gangliosides and α-synuclein in vitro.

FIG. 2A is a representation of a Western Blot, of α-synuclein proteinexpressed in MES23.5 cells transiently transfected with indicated levelsof an α-synuclein cDNA plasmid.

FIG. 2B is a representation of a Western Blot indicating the effects ofGBA cDNA transfection, +/−prosaposin cDNA transfection on theintracellular α-synuclein protein levels in MES23.5-syn cells. Thelevels of the latter protein are shown in the lower panel of the Westernblot.

FIG. 2C is a bar graph of ELISA measurements indicating theintra-cellular α-synuclein protein levels detected in MES-syn cells inpresence or absence of prosaposin and dependent on the amount ofco-transfected GBA DNA.

FIG. 3A is a graph of the performance of a sandwich ELISA thatspecifically detects increasing amounts of recombinant α-synucleinprotein (x-axis), as monitored by OD absorbance reading (y-axis).

FIG. 3B is a bar graph of ELISA measurements indicating theintra-cellular α-synuclein protein levels detected in MES23.5-syn cellsin the presence of increasing amounts of SNCA (synuclein, alpha (non A4component of amyloid precursor)) cDNA (x-axis) that was transfected intothese cells, where lysates were diluted in a serial manner from 1 in 200to 1 in 4,000.

FIG. 3C is a regression analysis of the concentration of intracellularα-synuclein protein after a given amount of transfected SCNA cDNA, asmeasured by sandwich ELISA, and as interpolated from the data obtainedin FIG. 3A and FIG. 3B.

FIG. 3D is a bar graph showing the results of lactate dehydrogenase(LDH) and MTT assays confirming full cellular viability of theα-synuclein protein expressing MES-syn cells, 24 hours aftertransfection.

FIG. 4 is a representation of sandwich ELISA results from five separateexperiments, which indicate a 20 to >270 percent rise in intracellularα-synuclein protein levels in MES23.5 cells following the expression ofmutant GBA polypeptides that carry one of several missense mutationsthat were recently linked to Parkinson disease and dementia with Lewybodies, or which carry mutations detrimental to the GBA active site. Theα-synuclein protein level is expressed in relation to the concentrationof MES-syn cells transfected with no ectopic GBA, but empty vector cDNAonly.

FIG. 5 is a bar graph of sandwich ELISA measurements of human and ratα-synuclein protein level-lowering activity of human cathepsin D whenjointly expressed in MES23.5-syn cells (MES-αS).

FIG. 6 is a representation of a Western Blot indicating thedose-dependent α-synuclein protein level-lowering activity of humancathepsin D when jointly expressed in MES23.5-syn cells (MES-αS).

FIG. 7 is a bar graph of sandwich ELISA measurements of the humanα-synuclein protein level-lowering activity of cathepsin D when jointlyexpressed in MES23.5-syn cells (MES-αS). Note, both wild-typeα-synuclein protein can be reduced by cathepsin D as well as severalmutant α-synuclein isoforms that carry missense mutations and have beenpreviously linked to familial forms of the disease and toautopsy-confirmed Parkinson's.

DETAILED DESCRIPTION

In general, the invention relates to methods of reducing the levels ofαS in cells in human or animal subjects who have a synucleinopathy thatis not a lysosomal storage disorder, e.g., subjects who have primary (orinvariable) synucleinopathy. These methods include, for example,administering non-protease-type lysosomal enzyme polypeptides, such asGBA polypeptides, or protease-type lysosomal enzyme polypeptides, suchas cathepsin D polypeptides, or nucleic acid molecules that encode suchpolypeptides, either alone or in combination with agents that enhance orinduce autophagy, such as rapamycin or a rapamycin analog. In addition,other GBA-activating agents, such as prosaposin polypeptides and/orsaposin C polypeptides can be administered, or prosaposin polypeptidesand/or saposin C polypeptides can be administered alone (to enhanceactivation of endogenous GBA activity) to facilitate a reduction in αSsteady-state protein levels in vivo.

Thus, the present invention involves modulating the physiologicaldegradation of α-synuclein (αS) by enhancing the processing within thelysosomes and/or the cytoplasm, and, in some embodiments, by enhancingthe amount of αS taken up by the lysosomes (autophagy). While notwishing to be bound by any theory of operation, the degradativeprocessing of αS aggregates is a system functioning at a steady-statelevel. By applying the law of mass action to the steady statedegradative processing of αS proteins, oligomeric forms, aggregates,and/or complexes, one can modulate the degradative process towards itsend products by increasing the abundance of its educts. By altering thecomponent reactions of the pathway one can push the overall processingtoward higher product levels. Thus, by either increasing the input of αSinto lysosomes or enhancing the degradation efficiency itself, one canpush the hydrolysis and subsequent processing of αS to a higher productlevel. Increasing both the autophagic component and the lysosomalcomponent of the pathway can lead to increased protection from αSprotein damage, and such combinations can achieve greater than additiveeffects.

Some embodiments described herein are methods of treating or delayingthe progression or development of a synucleinopathy disorder that is nota lysosomal storage disease, e.g., by administering an agent or agentsthat increases the activity or level of GBA and/or prosaposin/saposin C.In some embodiments, the agents can be GBA and/or prosaposin/saposin Cpolypeptides or active fragments thereof, or nucleic acids encoding suchpolypeptides or active fragments. In some embodiments, the agent is abinding-competent, but catalytically inactive form of GBA, or a nucleicacid molecule encoding the same.

Other embodiments described herein are methods of treating or delayingthe progression or development of a synucleinopathy disorder, e.g., byadministering an agent or agents that increases the activity or level ofcathepsin D or cathepsin F and/or preprocathepsin D or preprocathepsinF. In some embodiments, the agents can be GBA and/or prosaposin/saposinC/cathepsin D/cathepsin F polypeptides or active fragments thereof, ornucleic acids encoding such polypeptides or active fragments. In someembodiments, the agent is a binding-competent, but catalyticallyinactive form of GBA, cathepsin D or cathepsin F, or a nucleic acidmolecule encoding the same.

Methods of Treating Synucleinopathy Disorders

The term synucleinopathy is used herein to name a group ofneurodegenerative disorders characterized by the presence of increasedlevels, e.g., steady-state levels, of any one or more of solublenon-fibrillary variants, soluble oligomeric isoforms, insolublenon-fibrillary variants, complexes, and insoluble fibrillary aggregatesof α-synuclein (αS) protein within cellular compartments of selectivepopulations of neurons and glia. Thus, the αS steady-state level isunderstood to encompass all soluble as well as insoluble andintermediate (metastable) forms of the SNCA gene product.

These disorders include any one of the following grouped as “invariable”(or “primary”) synucleinopathies (Schlossmacher M G. a—synuclein andsynucleinopathies. The Dementias 2 Blue Books of Practical Neurology;Editors: Growdon J H & Rossor M N. Butterworth Heinemann, Inc., Oxford.2007; Chapter 8: pp 184-213): Parkinson's disease (PD) e.g., sporadicParkinson disease/parkinsonism and familial Parkinsondisease/parkinsonism; sporadic or heritable dementia with Lewy bodies(DLB) (aka diffuse Lewy body disease); pure autonomic failure (PAF) withsynuclein deposition; multiple system atrophy (MSA) (of cerebellar,parkinsonian, or mixed type); hereditary neurodegeneration with brainiron accumulation (aka, Hallervordern Spatz disease or pantothenatekinase 2-linked neurodegeneration); and incidental Lewy body disease ofadvanced age.

Furthermore, “variable” (or “secondary”) synucleinopathies have beenidentified, where dysregulation of the alpha-synuclein metabolism isrecognized to be a secondary event (given the abundance of the proteinin the nervous system), which nevertheless contributes significantly tothe course, penetrance, age-of-onset, severity and expressivity of theprimary illness. Disorders with variable synucleinopathy (SchlossmacherM G. a—synuclein and synucleinopathies. The Dementias 2 Blue Books ofPractical Neurology; Editors: Growdon J H & Rossor M N. ButterworthHeinemann, Inc., Oxford. 2007; Chapter 8: pp 184-213) include, but arenot limited to, Alzheimer's disease of the Lewy body variant; Down'ssyndrome; progressive supranuclear palsy; essential tremor with Lewybodies; familial parkinsonism with or without dementia resulting from amutant gene and loci where no gene mutation has yet been identified;Creutzfeldt Jakob disease and related prion diseases such as bovinespongiform encephalopathy (mad cow disease); secondary Parkinsondisease/parkinsonism resulting from neurotoxin exposure/drug-inducedparkinsonism with α-synuclein deposition; sporadic or heritablespinocerebellar ataxia; amyotrophic lateral sclerosis (ALS); idiopathicrapid eye movement sleep behavior disorder; and other conditionsassociated with central and/or peripheral α-synuclein accumulation inmammals accompanying a primary disease process.

Clinically, all of these related disorders are characterized by achronic and progressive decline in motor, cognitive, behavioral, and/orautonomic functions, depending on the distribution of thealpha-synuclein abnormalities.

A synucleinopathy may or may not be associated with disease symptoms. Itmay also be the product of normal aging. For example, persons over 55,60, 65, 70, 75, or 80, may accumulate such αS proteins, e.g., in theform of aggregates, without obvious association with a pathology,symptom, or disease state. This condition is referred to as incidentalLewy body disease (see above) and people with this condition areconsidered to be at higher risk for PD/parkinsonism.

In general, subjects with the types of synucleinopathies contemplatedherein do not have a clinically diagnosed (or not clinicallydiagnosable) primary lysosomal storage disorder (LSD), such as Gaucherdisease or Tay-Sachs disease; these LSD syndromes often demonstrate anautosomal recessive inheritance pattern. However, subjects with singleallele mutations in a gene that has been otherwise linked to a classicalLSD phenotype may also develop synucleinopathy and suffer from itsconsequences (such as PD/parkinsonism or dementia with Lewy bodies), butwithout evidence of a systemic LSD (Eblan et al., N. Engl. J. Med.,2005).

LSDs are a group of metabolic disorders including over forty geneticdisorders, many of which involve genetic defects in various lysosomalhydrolases that are commonly caused by mutations in both alleles of thegene that codes for the lysosomal enzyme. The hallmark feature of LSDsis the loss of 90 percent (or more) in enzymatic activity of thelysosomal hydrolase in question and the resulting abnormal accumulationof metabolites within lysosomes, which leads to the formation of largenumbers of distended lysosomes in the perikaryon.

The methods described herein can be used to treat all persons withprimary or secondary synucleinopathies, including those withoutmutations in their lysosomal enzyme genes, such as the GBA gene (i.e.,sporadic Parkinson disease patients without a known single geneabnormality). These are patients where aging/toxic insult/headtrauma/influence of modifier genes or other unknown causes may worktogether to cause or promote disease (Klein and Schlossmacher,Neurology, 2007, 69(22):2093-104).

The new methods described herein can also be used to treat asub-population of synucleinopathy patients with a heterozygous (i.e.,single allele rather than two allele) mutation in one or more of thelysosomal enzyme genes, e.g., in the GBA gene. These subjects are notsuffering from a typical LSD (because they do not have a 90 percent orgreater enzyme deficiency, because they still express enough GBA fromthe one remaining healthy allele), but they often suffer from a primarysynucleinopathy. The currently available data suggest that thisheterozygous mutation in GBA serves as a risk factor for Parkinsondisease (and related disorders) and as a risk allele for the developmentof a primary synucleinopathy in the nervous system (Clark et al.,Neurology, 2007, 69(12):1270-7). Intriguingly, if both copies (alleles)of the GBA gene are mutated (for example in carriers of N370S, L444P,K198T, and R329c variants of GBA), a subgroup of patients with theclassical LSD features of Gaucher disease will develop secondarysynucleinopathy (Lwin et al., Mol. Genet. Metab., 2004, 81(1):70-3).

In particular, the treatments can be applied prophylactically to thosepeople who are genotyped for known GBA mutations (who, for example, havealready been genotyped due to a family history of Gaucher Disease) toprevent the development of Parkinson disease or therapeutically in thosepeople with known GBA mutations who have already developed asynucleinopathy disorder. Thus, the step of genotyping the patient orsubject for a mutation in a lysosomal enzyme gene, e.g., the GBA gene,can be a first step in the therapeutic methods described herein.Patients who are heterozygotes for the mutation, are candidates fortreatment by the new methods.

Administering or Activating Non-Protease-Type Lysosomal EnzymePolypeptides

The new methods include administering non-protease-type lysosomal enzymepolypeptides, e.g., GBA polypeptides, either directly, or byadministering nucleic acid molecules that encode GBA polypeptides, topatients in need thereof, e.g., in subjects having been diagnosed with asynucleinopathy that is not a lysosomal storage disorder.

GBA is also known as glucosidase, beta, acid; acid beta-glucosidase;acid beta-glucosidase; glucocerebrosidase; glucosylceramidase; and GBAP.This gene normally encodes a lysosomal membrane protein that cleaves thebeta-glucosidic linkage of glucosylceramide (also known asglucocerebroside), an intermediate in glycolipid metabolism. It can alsocleave glucosylsphingosine as a secondary substrate to generate glucoseand sphingosine (Sidransky, Mol Genet Met, 2004, pp 6-15). Mutations inthis gene can cause Gaucher disease, a lysosomal storage diseasecharacterized by an accumulation of glucocerebrosides andglucosylsphingosines. Alternative splicing results in multipletranscript variants encoding the same protein. There are five mRNAvariants (which vary in the 5′ UTR), the longest of which is set forthin the GenBank database at Accession Nos. NM_(—)001005749.1 (mRNA) andNP_(—)001005749.1 (amino acid). Information regarding GBA can be foundin the Entrez Gene database at GeneID: 2629.

The methods can also include increasing activation of the administeredGBA polypeptides or any endogenous GBA by administering GBA-activatingpolypeptides, such as prosaposin (PS) and/or its derivatives, saposin A(SA), saposin B (SB), saposin C (SC), and saposin D (SD).

The prosaposin gene encodes a highly conserved glycoprotein which iseither secreted as a full length protein with neurotrophic activities,or proteolytically processed in endosomal/lysosomal compartments bycathepsin D and other proteases into the 4 saposins A, B, C and D(Leonova et al., J. Biol. Chem., 1996, 271:17312-17320; Hiraiwa et al.,Arch. Biochem. Biophys., 1997, 341:17-24). Saposins A-D localizeprimarily to the lysosomal compartment where they facilitate thecatabolism of glycosphingolipids with short oligosaccharide groups.Saposin C functions to anchor the GBA protein under low pH conditions tothe internal side of the lysosomal membrane, thus allowing GBA to foldproperly for correct substrate interaction (Salvioli et al., 2000, FEBS.Lett. 472:17-21). Furthermore, saposin C protects the GBA protein fromproteolytic degradation by lysosomal proteases (Sun et al., J. Biol.Chem., 2003, 278:31918-31923). The biological importance of this proteinis underscored by the fact that null mutations in the prosaposin geneand/or point mutations in the Saposin C region of the gene can lead toclinical Gaucher Disease, despite the presence of wild type GBA (Pamploset al., Acta. Neuropathol., 1999, 97:91-97; Tylki-Szymanska, 2007, Clin.Genet., 72:538-542; Rafi et al., 1993, Somat. Cell Mol. Genet., 19:1-7).

Furthermore, the low activity in vivo of the most common GBA mutation,N370S, can be accounted for by its inability to interact with saposin Cand anionic phospholipids (Salvioli et al., Biochem. J., 2005,390:95-103). Alternative splicing of prosaposin results in multipletranscript variants encoding different isoforms. Prosaposin (variantGaucher disease and variant metachromatic leukodystrophy) is describedin the Entre Gene database at GeneID: 5660. The sequences of itsisoforms are available in GenBank as follows: prosaposin isoform apreproprotein: NM_(—)002778.2 (mRNA) and NP_(—)002769.1 (amino acid);prosaposin isoform b preproprotein: NM_(—)001042465.1 (mRNA) andNP_(—)001035930.1 (amino acid); and prosaposin isoform c preproproteinNM_(—)001042466.1 (amino acid) and NP_(—)001035931.1 042465.1 (mRNA).

Both the GBA polypeptides and GBA encoding nucleic acid molecules, aswell as the GBA-activating polypeptides and corresponding nucleic acidmolecules, can be administered using known techniques, including thetechniques described herein. For example, the GBA polypeptide encodingnucleic acid molecules can be administered using gene therapy asdescribed herein.

Administering Protease-Type Lysosomal Enzyme Polypeptides

In an alternative method, a subject diagnosed as having asynucleinopathy that is not a LSD, can be treated with a lysosomalprotease polypeptides, such as a cathepsin D polypeptide. Such proteasescan be administered directly, or by administering a nucleic acidmolecule that encodes the desired protease.

In general, protease-type lysosomal enzymes fall into the categories ofaspartyl proteases such as a cathepsin D (or cathepsin E), and cysteinylproteases (e.g., cathepsin F and cathepsin L). Therefore, the inventionincludes, inter alia, new methods of treating synucleinopathies withprotease-type lysosomal enzymes related to procathepsin D orprocathepsin E polypeptide, or alternatively with protease-typelysosomal enzymes related to procathepsin F or procathepsin Lpolypeptide, or nucleic acid molecules that encode a cathepsin D,cathepsin E, cathepsin F, or cathepsin L, or those that encode theirpre-pro-protein polypeptide forms.

The cathepsin family of proteases includes approximately a dozenmembers, which are distinguished by their structure and the proteinsthey cleave. Most of the members become activated at the low pH found inlysosomes. Thus, the activity of this family lies almost entirely withinthose organelles. The cathepsin D gene (CTSD) encodes a lysosomalaspartyl protease composed of a dimer of disulfide-linked heavy andlight chains, both produced from a single protein precursor. Thisproteinase, which is a member of the peptidase C1 family, has aspecificity similar to, but narrower than, that of pepsin A. Sequenceinformation of the human gene is available in GenBank as Homo sapienscathepsin D (CTSD), mRNA: NM_(—)001909.

Within the cathepsin family, only one other known member (besidescathepsin D) possesses aspartyl protease activity, that is cathepsin E.It is transcribed in 2 variants. Sequence information for the humanvariants is available in GenBank as Homo sapiens cathepsin E (CTSE),mRNA: NM_(—)001910.2 and NM_(—)148964.1.

Within the cathepsin family, various other members possess cysteineprotease activity, for example cathepsins C, L, F and W. Of these manycysteine protease cathepsins, the F and W enzymes form a separatesubgroup, based on their chromosomal locations, sequence homology andsplicing pattern (Wex et al., 1999, Biochem. Biophys. Res. Commun.,259:401-407). Cathepsin F is expressed in brain, as well as heart,skeletal muscle and other tissues (Wang et al., 1998, J. Biol. Chem.,273:32000-32008). Knock out of the cathepsin F gene in mice leads to alate onset neurological disease with gliosis, neuronal loss andaccumulation of autofluorescent granules (Tang et al., 2006, Mol. Cell.Biol., 26:2309-2316), which is thought to be a model of humanadult-onset neuronal ceroid lipofuscinosis. Sequence information of thehuman gene is available in GenBank as Homo sapiens cathepsin E (CTSE),mRNA: NM_(—)003793.3.

Both the cathepsin D or F polypeptides and cathepsin D or F encodingnucleic acid molecules can be administered using known techniques,including the techniques described herein. For example, the cathepsin Dencoding nucleic acid molecules can be administered using gene therapyas described herein.

Administering Other Lysosomal Enzyme Polypeptides

Examples of other polypeptides that can be administered to enhance thedegradative processing of αS within lysosomes includeAspartylglucosaminidase; α-Galactosidase A; Palmitoyl ProteinThioesterase; Tripeptidyl Peptidase; Lysosomal Transmembrane Protein;Cysteine transporter; Acid ceramidase; Acid α-L-fucosidase; Protectiveprotein/cathepsin A; Acid β-galactosidase; Iduronate-2-sulfatase;α-L-Iduronidase; Galactocerebrosidase; Acid α-mannosidase; Acidβ-mannosidase; Arylsulfatase B; Arylsulfatase A;N-Acetylgalactosamine-6-sulfate; Acid β-galactosidase;N-Acetylglucosamine-1-phosphotransferase; Acid sphingomyelinase; NPC-1;α-glucosidase; β-Hexosaminidase B; Heparan N-sulfatase;α-N-Acetylglucosaminidase; Acetyl-CoA: α-glucosaminide;N-Acetylglucosamine-6-sulfate; α-N-Acetylgalactosaminidase;α-N-Acetylgalactosaminidase; α-Neuramidase; β-Glucuronidase;β-Hexosaminidase A; glucocerebrosidase; ubiquitin C-terminalhydrolase-L1; and Acid Lipase.

The proteins and polypeptides can be lysosomal degradation enzymes ornon-lysosomal proteins that promote degradation of synuclein orsynuclein aggregates. These proteins and their coding sequences are wellknown in the art. Typically the human forms of the proteins and theircoding sequences will be used, although for work in animal models, theanimal orthologs may be desirable. One or more of such enzymes can beused.

Enhancing and Inducing Autophagy of Alpha-Synuclein

In another aspect of the invention, agents that enhance and/or induceautophagy, such as rapamycin or rapamycin analogs are co-administeredwith lysosomal enzymes, e.g., GBA polypeptides, or non-GBA-typelysosomal proteases, such as cathepsin D polypeptides, to achieve agreater than additive therapeutic effect.

Autophagy is a catabolic process involving the degradation of a cell'sown components through the lysosomal machinery. It is atightly-regulated process that plays a normal part in cell growth,development, and homeostasis, helping to maintain a balance between thesynthesis, degradation, and subsequent recycling of cellular products.It is a major mechanism by which a starving cell reallocates nutrientsfrom unnecessary processes to more-essential processes.

A variety of autophagic processes exist, all having in common thedegradation of intracellular components via the lysosome. The mostwell-known mechanism of autophagy involves the formation of a membranearound a targeted region of the cell, separating the contents from therest of the cytoplasm. The resultant vesicle then fuses with a lysosomeand subsequently degrades the contents.

Autophagy can be broadly separated into three types: macroautophagy,microautophagy, and chaperone-mediated autophagy: (i) macroautophagyinvolves the formation of a de-novo-formed membrane sealing on itself toengulf cytosolic components (proteins and/or whole organelles), whichare degraded after its fusion with the lysosome; (ii) microautophagy isthe direct invagination of materials into the lysosome; and (iii)chaperone-mediated autophagy (CMA) involves the degradation of specificcytosolic proteins marked with a specific peptide sequence. CMA is veryselective in what is degraded and degrades only certain proteins and notorganelles. CMA is responsible for the degradation of approximately 30%of cytosolic proteins in tissues such as liver, kidney and in many typesof cultured cells.

Chaperone molecules bind to and transport marked proteins to thelysosome via a receptor complex. In CMA, only those proteins that have aconsensus peptide sequence get recognized by the binding of a chaperone.This CMA substrate/chaperone complex then moves to the lysosomes, wherea CMA receptor lysosome-associated membrane protein recognizes thecomplex; the protein is unfolded and translocated across the lysosomemembrane assisted by additional proteins on the inside. Solublewild-type α-synuclein has been reported to be degraded by this mechanism(Cuervo et al. (2004), Science, 305:1292).

Autophagy is part of everyday normal cell growth and development whereinthe mammalian target of rapamycin (mTOR) plays an important regulatoryrole. Starvation inhibits mTOR activity, provoking various cellularresponses, including cell arrest in the early G1 phase, inhibition ofprotein synthesis, nutrient transporter turnover, transcriptionalchanges, and autophagy. Rapamycin is a well known agent for theinhibition of mTOR activity. Any rapamycin analog or mTOR inhibitorknown in the art can be used for the methods described herein. Forexample, everolimus, cyclosporine, and FK506 can be used or tested fortheir autophagy stimulatory capacity. Although all of such analogs andinhibitors may not have the autophagy stimulatory activity of rapamycin,this activity can be readily determined among these compounds. Theagents that promote autophagy include chaperone proteins and compoundsthat bind to and escort substrates to the lysosome. Other compounds thatcan stimulate autophagy include hsc70, N-octyl-4-epi-β-valienamine, andglycerol. One or more of such agents can be used in the methods ofenhancing autophagy described herein.

Any lysosomal enzyme that helps to degrade synuclein or causedisaggregation of synuclein complexes, alone or in combination withother lysosomal enzyme(s) or agent(s), can be used for the presentinvention.

Methods of Administration

Generally, the methods described herein include administering atherapeutically effective amount of a therapeutic compound as describedherein, to a subject who is in need of, or who has been determined to bein need of, such treatment.

As used in this context, to “treat” means to ameliorate at least onesymptom of the synucleinopathy disorder and/or to cause a measurabledecrease in the level of αS protein in the subject. Similarly,administration of a “therapeutically effective amount” or “effectiveamount” of a composition described herein for the treatment of asynucleinopathy will result in a decreased level of αS protein and/orresults in an improvement in one or more symptoms of the synucleinopathydisorder. This amount can be the same or different from a“prophylactically effective amount,” which is an amount necessary toinhibit, e.g., prevent, onset of disease or disease symptoms.

An effective amount can be administered in one or more administrations,applications, or dosages. A therapeutically effective amount of acomposition depends on the composition selected. The compositions can beadministered from one or more times per day to one or more times perweek; including once every other day. The skilled artisan willappreciate that certain factors influence the dosage and timing requiredto effectively treat a subject, including, but not limited to, theseverity of the disease or disorder, previous treatments, the generalhealth and/or age of the subject, and other diseases present. Moreover,treatment of a subject with a therapeutically effective amount of thecompositions described herein can include a single treatment or a seriesof treatments.

Dosage, toxicity, and therapeutic efficacy of the compounds can bedetermined, e.g., by standard pharmaceutical procedures in cell culturesor experimental animals, e.g., for determining the LD50 (the dose lethalto 50% of the population) and the ED50 (the dose therapeuticallyeffective in 50% of the population). The dose ratio between toxic andtherapeutic effects is the therapeutic index and it can be expressed asthe ratio LD50/ED50. Compounds that exhibit high therapeutic indices arepreferred. While compounds that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

More specific information on dosages is discussed below.

Administration of Polypeptides and Small Molecules

Agents that cross the blood-brain barrier can be administeredsystemically, if desired. Alternatively, agents such as the polypeptidesand small molecules described herein, can be delivered directly to asite in the body where cells display αS accumulation. Such agents thatdo not cross the blood brain barrier can be administered to the brain,for example, using direct injection facilitated by stereotacticguidance. Such agents can also be administered via intraventricular orintraparenchymal routes.

In other embodiments, nucleic acid molecules encoding the desiredpolypeptides can be delivered, e.g., in the form of a viral vectorcontaining a polypeptide-encoding gene. The viral delivery may be underconditions that favor expression of the transgene in specific central orperipheral nerve cells, such as ependymal or other glial cells that linethe ventricles of the brain. Ependymal cells can be transduced toexpress the transgene and secrete the encoded protein product into thecerebrospinal fluid (CSF).

The polypeptides described herein can be incorporated into apharmaceutical composition useful to treat, e.g., inhibit, attenuate,prevent, or ameliorate, a synucleinopathy. The pharmaceuticalcomposition can be administered to a subject suffering from asynucleinopathy disorder or someone who is at risk of developing saiddeficiency. The compositions should contain a therapeutic orprophylactic amount of the polypeptide, in a pharmaceutically-acceptablecarrier. The pharmaceutical carrier can be any compatible, non-toxicsubstance suitable to deliver the polypeptides to the patient. Sterilewater, alcohol, fats, and waxes may be used as the carrier.Pharmaceutically-acceptable adjuvants, buffering agents, dispersingagents, and the like, may also be incorporated into the pharmaceuticalcompositions. The carrier can be combined with the polypeptide in anyform suitable for administration by intraventricular injection orinfusion (which form can also be suitable for intravenous or intrathecaladministration) or otherwise.

Suitable carriers include, for example, physiological saline,bacteriostatic water, Cremophor EL® (BASF, Parsippany, N.J.) orphosphate buffered saline (PBS), other saline solutions, dextrosesolutions, glycerol solutions, water and oils emulsions such as thosemade with oils of petroleum, animal, vegetable, or synthetic origin(peanut oil, soybean oil, mineral oil, or sesame oil). In someembodiments, an artificial CSF is used as a carrier. In general, thecarrier will be sterile and free of pyrogens. The concentration of thepolypeptide in the pharmaceutical composition can vary widely, i.e.,from at least about 0.01% by weight, to 0.1% by weight, to about 1%weight, to as much as 20% by weight or more of the total composition.

For intraventricular administration of the polypeptides describedherein, or other agents, the composition must be sterile and should be afluid. It must be stable under the conditions of manufacture and storageand must be preserved against the contaminating action of microorganismssuch as bacteria and fungi. Prevention of the action of microorganismscan be achieved by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, andthe like. In many cases, it will be useful to include isotonic agents,for example, sugars, polyalcohols such as mannitol, sorbitol, and sodiumchloride in the composition.

The rate of administration is such that the administration of a singledose can be administered as a bolus. A single dose can also be infusedover about 1-5 minutes, about 5-10 minutes, about 10-30 minutes, about30-60 minutes, about 1-4 hours, or consumes more than four, five, six,seven, or eight hours. It may take more than 1 minute, more than 2minutes, more than 5 minutes, more than 10 minutes, more than 20minutes, more than 30 minutes, more than 1 hour, more than 2 hours, ormore than 3 hours. While bolus intraventricular administrations areeffective, slow infusions are particularly effective. Without beingbound by any particular theory of operation, it is believed that theslow infusion is effective due to the turn-over of the CSF.

While estimates and calculations in the literature vary, the CSF isbelieved to turn over within about 4, 5, 6, 7, or 8 hours in humans. Inone embodiment, the slow infusion time should be metered so that it isabout equal to or greater than the turn-over time of the CSF. Turn-overtime may depend on the species, size, and age of the subject, but can bedetermined using methods known in the art. The infusion may also becontinuous over a period of one or more days. The patient can be treatedonce, twice, or three or more times a month, e.g., weekly, e.g., everytwo weeks. Infusions can be repeated over the course of a subject's lifeas dictated by re-accumulation of the disease's substrate in the brainor visceral organs. Re-accumulation can be determined by any of thetechniques that are well known in the art for the identification andquantization of the relevant substrate, which techniques may beperformed on one or more samples taken from the brain and/or from one ormore of the visceral organs. Such techniques include enzymatic assaysand/or immunoassays, e.g., radioimmunoassays or ELISAs.

Slow intraventricular infusion provides diminished amounts of thesubstrate for an administered polypeptide (e.g., an enzyme) in at leastthe brain and potentially in visceral organs. The reduction in asubstrate such as aS protein accumulated in the brain, lungs, spleen,kidney, and/or liver may be dramatic. Reductions of greater that 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% can be achieved. The reductionachieved is not necessarily uniform from patient to patient or even fromorgan to organ within a single patient. Reductions can be determined byany of the techniques that are well known in the art, e.g., by enzymaticassays and/or immunoassay techniques, as discussed elsewhere herein.

In an illustrative embodiment, the administration is accomplished byinfusion of the polypeptide into one or both of the lateral ventriclesof a subject or patient. By infusing into the lateral ventricles, thepolypeptide is delivered to the site in the brain in which the greatestamount of CSF is produced. The polypeptide can also be infused into morethan one ventricle of the brain. Treatment can consist of a singleinfusion per target site, or can be repeated. Multipleinfusion/injection sites can be used. For example, the ventricles intowhich the polypeptide is administered can include the lateral ventriclesand the fourth ventricle. In some embodiments, in addition to the firstadministration site, a composition containing the polypeptide isadministered to another site which can be contralateral or ipsilateralto the first administration site. Injections/infusions can be single ormultiple, unilateral or bilateral.

To deliver the solution or other composition containing the polypeptidespecifically to a particular region of the central nervous system, suchas to a particular ventricle, e.g., to the lateral ventricles or to thefourth ventricle of the brain, it can be administered by stereotaxicmicroinjection. For example, on the day of surgery, patients have astereotaxic frame base fixed in place (screwed into the skull). Thebrain with stereotaxic frame base (MRI compatible with fiduciarymarkings) is imaged using high resolution MRI. The MRI images are thentransferred to a computer that runs stereotaxic software. A series ofcoronal, sagittal, and axial images used to determine the target site ofvector injection, and trajectory. The software directly translates thetrajectory into 3-dimensional coordinates appropriate for thestereotaxic frame. Burr holes are drilled above the entry site and thestereotaxic apparatus localized with the needle implanted at the givendepth. The polypeptide solution in a pharmaceutically acceptable carrieris then injected. Additional routes of administration can be used, e.g.,superficial cortical application under direct visualization, or othernon stereotaxic application.

One way to deliver a slow infusion is to use a pump. Such pumps arecommercially available, for example, from Alzet (Cupertino, Calif.) orMedtronic (Minneapolis, Minn.). The pump may be implantable. Anotherconvenient way to administer the enzymes is to use a cannula or acatheter. The cannula or catheter can be used for multipleadministrations separated in time. Cannulae and catheters can beimplanted stereotaxically. It is contemplated that multipleadministrations will be used to treat the typical patient with asynucleinopathy disorder. Catheters and pumps can be used separately orin combination.

Administration of Nucleic Acid Molecules and Gene Therapy

The nucleic acid molecules described herein, such as nucleic acidmolecules encoding GBA or cathepsin D polypeptides, can be deliveredusing a number of different methods. For example, gene transfer can bemediated by a DNA viral vector, such as an adenovirus (Ad) oradeno-associated virus (AAV). A vector construct refers to apolynucleotide molecule including the viral genome or part thereof and atransgene. Adenoviruses (Ads) are a relatively well characterized,homogenous group of viruses, including over 50 serotypes. See, e.g.,International PCT Application No. WO 95/27071. Ads are easy to grow anddo not require integration into the host cell genome. Recombinant Adderived vectors, particularly those that reduce the potential forrecombination and generation of wild-type virus, have also beenconstructed. See, International PCT Application Nos. WO 95/00655 and WO95/11984. Wild-type AAV has high infectivity and specificity integratinginto the host cell's genome. See, Hermonat and Muzyczka (1984) Proc.Natl. Acad. Sci., USA, 81:6466-6470 and Lebkowski et al. (1988) Mol.Cell. Biol., 8:3988-3996.

Suitable neurotrophic viral vectors to deliver the nucleic acidmolecules described herein include, but are not limited to,adeno-associated viral vectors (AAV), herpes simplex viral vectors (U.S.Pat. No. 5,672,344) and lentiviral vectors.

In the new methods, AAV of any serotype or pseudotype can be used. Theserotype of the viral vector used in certain embodiments of theinvention is selected from the group consisting from AAV1, AAV2, AAV3,AAV4, AAV5, AAV6, AAV7, and AAV8 (see, e.g., Gao et al. (2002) PNAS,99:11854 11859; and Viral Vectors for Gene Therapy: Methods andProtocols, ed. Machida, Humana Press, 2003). Other serotype besidesthose listed herein can be used. Furthermore, pseudotyped AAV vectorscan also be utilized in the methods described herein. Pseudotyped AAVvectors are those that contain the genome of one AAV serotype in thecapsid of a second AAV serotype; for example, an AAV vector thatcontains the AAV2 capsid and the AAV1 genome or an AAV vector thatcontains the AAV5 capsid and the AAV 2 genome (Auricchio et al., (2001)Hum. Mol. Genet., 10(26):3075-81).

AAV vectors are derived from single stranded (ss) DNA parvoviruses thatare nonpathogenic for mammals (reviewed in Muzyscka (1992) Curr. Top.Microb. Immunol., 158:97-129). Briefly, AAV based vectors have rep andcap viral genes that account for 96% of the viral genome removed,leaving the two flanking 145 basepair (bp) inverted terminal repeats(ITRs), which are used to initiate viral DNA replication, packaging andintegration. In the absence of helper virus, wild type AAV integratesinto the human host cell genome with preferential site specificity atchromosome 19q13.3 or it can be maintained episomally. A single AAVparticle can accommodate up to 5 kb of ssDNA, therefore leaving about4.5 kb for a transgene and regulatory elements, which is typicallysufficient. However, trans-splicing systems as described, for example,in U.S. Pat. No. 6,544,785, may nearly double this limit.

In an illustrative embodiment, AAV is AAV2. Adeno associated virus ofmany serotypes, especially AAV2, have been extensively studied andcharacterized as gene therapy vectors. Those skilled in the art arefamiliar with the preparation of functional AAV-based gene therapyvectors. Numerous references to various methods of AAV production,purification, and preparation for administration to human subjects canbe found in the extensive body of published literature (see, e.g., ViralVectors for Gene Therapy: Methods and Protocols, ed. Machida, HumanaPress, 2003). Additionally, AAV based gene therapy targeted to cells ofthe CNS has been described in U.S. Pat. Nos. 6,180,613 and 6,503,888.Additional exemplary AAV vectors are recombinant AAV2/1, AAV2/2, AAV2/5,AAV2/6, AAV2/7, and AAV2/8 serotype vectors encoding human protein.

In certain methods described herein, the vector includes a transgeneoperably linked to a promoter. The transgene encodes a biologicallyactive molecule, such as a GBA polypeptide, expression of which in theCNS results in at least partial correction of a synucleinopathy.

The level of transgene expression in eukaryotic cells is largelydetermined by the transcriptional promoter within the transgeneexpression cassette. Promoters that show long term activity and aretissue- and even cell-specific are used in some embodiments. Examples ofpromoters include, but are not limited to, the cytomegalovirus (CMV)promoter (Kaplitt et al. (1994) Nat. Genet., 8:148-154), CMV/human β3globin promoter (Mandel et al. (1998) J. Neurosci., 18:4271-4284), GFAPpromoter (Xu et al. (2001) Gene Ther., 8:1323-1332), the 1.8 kb neuronspecific enolase (NSE) promoter (Klein et al. (1998) Exp. Neurol.,150:183-194), chicken beta actin (CBA) promoter (Miyazaki (1989) Gene,79:269-277), the β-glucuronidase (GUSB) promoter (Shipley et al. (1991)Genetics, 10:1009-1018), and ubiquitin promoters such as those isolatedfrom human ubiquitin A, human ubiquitin B, and human ubiquitin C, asdescribed in U.S. Pat. No. 6,667,174. To prolong expression, otherregulatory elements may additionally be operably linked to thetransgene, such as, e.g., the Woodchuck Hepatitis Virus Post RegulatoryElement (WPRE) (Donello et al. (1998) J. Virol., 72:5085-5092) or thebovine growth hormone (BGH) polyadenylation site.

For some CNS gene therapy applications, it may be necessary to controltranscriptional activity. To this end, pharmacological regulation ofgene expression with viral vectors can been obtained by includingvarious regulatory elements and drug responsive promoters as described,for example, in Haberma et al. (1998) Gene Ther., 5:1604-16011; and Yeet al. (1995) Science, 283:88-91.

High titer AAV preparations can be produced using techniques known inthe art, e.g., as described in U.S. Pat. No. 5,658,776 and Viral Vectorsfor Gene Therapy: Methods and Protocols, ed. Machida, Humana Press,2003.

Dosages

For the treatment of disease, the appropriate dosage of a polypeptide,e.g., a GBA or cathepsin polypeptide or other agent described herein,will depend on the type of disease to be treated, the severity andcourse of the disease, whether the polypeptide or agent is administeredfor prophylactic or therapeutic purposes, previous therapy, thepatient's clinical history and response to the enzyme or agent, and thediscretion of the attending physician.

In a combination therapy regimen, the compositions described herein areadministered in a therapeutically effective or synergistic amount. Atherapeutically synergistic amount is that amount of one or morepolypeptides or other agents in combination with one or more otherpolypeptides or agents, necessary to significantly reduce or eliminateconditions or symptoms associated with a particular disease in a mannerthat is more than additive when the two polypeptides/agents areadministered alone.

While dosages may vary depending on the disease and the patient, thepolypeptide is generally administered to the patient in amounts of fromabout 0.1 to about 1000 milligrams per 50 kg of patient eachadministration and may be repeated weekly, monthly, or at other timeintervals as needed. In one embodiment, the polypeptide is administeredto the patient in amounts of about 1 to about 500 milligrams per 50 kgof patient per month. In other embodiments, the polypeptide isadministered to the patient in amounts of about 5 to about 300milligrams per 50 kg of patient per month, or about 10 to about 200milligrams per 50 kg of patient per month.

Depending on the type and severity of the disease, the polypeptide oragent can be administered so that the local concentration provided isabout 100 pg/ml to about 100 μg/ml, 1 ng/ml to about 95 μg/ml, 10 ng/mlto about 85 μg/ml, 100 ng/ml to about 75 μg/ml, from about 100 ng/ml toabout 50 μg/ml, from about 1 μg/ml to about 25 μg/ml, from about 1 μg/mlto about 15 μg/ml, from about 1 μg/ml to about 10 μg/ml, or from about 1μg/ml to about 4 μg/ml.

When the polypeptide or agent is delivered by gene therapy through viralvirions, the dose can be from about 2×10⁶ to about 2×10¹² drp, fromabout 2×10⁷ to about 2×10¹¹ drp, or from about 2×10⁸ to about 2×10¹¹ drp(DNase resistant particles) per unit dose. In certain embodiments, theconcentration or titer of the vector in the composition is at least: (a)5, 6, 7, 8, 9, 10, 15, 20, 25, or 50 (×10¹² gp/ml); (b) 5, 6, 7, 8, 9,10, 15, 20, 25, or 50 (×10⁹ tu/ml); or (c) 5, 6, 7, 8, 9, 10, 15, 20,25, or 50 (×10¹⁰ iu/ml).

The terms “genome particles (gp),” or “genome equivalents,” as used inreference to a viral titer, refer to the number of virions containingthe recombinant AAV DNA genome, regardless of infectivity orfunctionality. The number of genome particles in a particular vectorpreparation can be measured by procedures such as described in theExamples herein, or for example, in Clark et al. (1999) Hum. Gene Ther.,10:1031-1039; Veldwijk et al. (2002) Mol. Ther., 6:272-278.

The terms “infection unit (iu),” “infectious particle,” or “replicationunit,” as used in reference to a viral titer, refer to the number ofinfectious and replication-competent recombinant AAV vector particles asmeasured by the infectious center assay, also known as replicationcenter assay, as described, for example, in McLaughlin et al. (1988) J.Virol., 62:1963-1973.

The term “transducing unit (tu)” as used in reference to a viral titer,refers to the number of infectious recombinant AAV vector particles thatresult in the production of a functional transgene product as measuredin functional assays such as described in Examples herein, or forexample, in Xiao et al. (1997) Exp. Neurobiol., 144:113-124; or inFisher et al. (1996) J. Virol., 70:520-532 (LFU assay).

When the polypeptide or agent is administered by protein or chemicaltherapy, the dose can be from about 0.1 mg to about 50 mg, from about0.1 mg to about 25 mg, from about 0.1 mg to about 10 mg, from about 0.5mg to about 5 mg, or from about 0.5 mg to about 2.5 mg per unit dose.

The polypeptides and agents described herein can be administered as asingle dose or repeatedly. For repeated administrations over severaldays or longer, depending on the condition, the treatment is sustaineduntil a desired suppression of disease symptoms occurs. The progress ofthe therapy of the invention is monitored by conventional techniques andassays.

Pharmaceutical Compositions

A “pharmaceutical composition” or “medicament” is intended to encompassa combination of an active component or agent, e.g., an enzymepolypeptide, and optionally a carrier or other material, e.g., acompound or composition, which is inert (for example, a detectable agentor label) or active, such as an adjuvant, diluent, binder, stabilizer,buffer, salt, lipophilic solvent, preservative, adjuvant or the like, ora mixture of two or more of these substances.

Carriers are preferably pharmaceutically acceptable. They may includepharmaceutical excipients and additives, proteins, peptides, aminoacids, lipids, and carbohydrates (e.g., sugars, includingmonosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatizedsugars such as alditols, aldonic acids, esterified sugars and the like;and polysaccharides or sugar polymers), which can be present singly orin combination, comprising alone or in combination 1-99.99% by weight orvolume. Exemplary protein excipients include serum albumin such as humanserum albumin (HSA), recombinant human albumin (rHA), gelatin, casein,and the like. Representative amino acid/antibody components, which canalso function in a buffering capacity, include alanine, glycine,arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine,lysine, leucine, isoleucine, valine, methionine, phenylalanine,aspartame, and the like. Carbohydrate excipients are also intendedwithin the scope of this invention, examples of which include but arenot limited to monosaccharides such as fructose, maltose, galactose,glucose, D-mannose, sorbose, and the like; disaccharides, such aslactose, sucrose, trehalose, cellobiose, and the like; polysaccharides,such as raffinose, melezitose, maltodextrins, dextrans, starches, andthe like; and alditols, such as mannitol, xylitol, maltitol, lactitol,xylitol sorbitol (glucitol) and myoinositol.

The term carrier also includes a buffer or a pH adjusting agent or acomposition containing the same; typically, the buffer is a saltprepared from an organic acid or base. Representative buffers includeorganic acid salts such as salts of citric acid, ascorbic acid, gluconicacid, carbonic acid, tartaric acid, succinic acid, acetic acid, orphthalic acid, Tris, tromethamine hydrochloride, or phosphate buffers.Additional carriers include polymeric excipients/additives such aspolyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g.,cyclodextrins, such as 2-hydroxypropyl-.quadrature.-cyclodextrin),polyethylene glycols, flavoring agents, antimicrobial agents,sweeteners, antioxidants, antistatic agents, surfactants (e.g.,polysorbates such as “TWEEN 20”® and “TWEEN 80”®), lipids (e.g.,phospholipids, fatty acids), steroids (e.g., cholesterol), and chelatingagents (e.g., EDTA).

As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutical carriers, such as saline,solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like, aphosphate buffered saline solution, water, and emulsions, such as anoil/water or water/oil emulsion, and various types of wetting agents,compatible with pharmaceutical administration. Supplementary activecompounds can also be incorporated into the compositions. Thecompositions and medicaments manufactured and/or used in accordance withthe present invention and which include the particular polypeptides,nucleic acid molecules or other agents can include stabilizers andpreservatives and any of the carriers described herein with theadditional proviso that they be acceptable for use in vivo. For examplesof additional carriers, stabilizers, and adjuvants, see MartinREMINGTON'S PHARM. SCI., 15th Ed. (Mack Publ. Co., Easton (1975) andWilliams & Williams, (1995), and in the “PHYSICIAN'S DESK REFERENCE,”52nd ed., Medical Economics, Montvale, N.J. (1998).

The methods described herein include the manufacture and use ofpharmaceutical compositions, which can include compounds identified bythe screening methods described herein as active ingredients. Alsoincluded are the pharmaceutical compositions themselves. For example,the compositions described herein can include agents that increase thelevel or activity of one or both of GBA or PS/SC.

Pharmaceutical compositions are typically formulated to be compatiblewith their intended route of administration. Examples of routes ofadministration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration.

Methods of formulating suitable pharmaceutical compositions are known inthe art, see, e.g., the books in the series Drugs and the PharmaceuticalSciences: a Series of Textbooks and Monographs (Dekker, N.Y.). Forexample, solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringes,or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injection can include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent that delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle, which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying, which yield a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel®, or corn starch; a lubricant such as magnesium stearate orSterotes®; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds can be delivered in theform of an aerosol spray from a pressured container or dispenser thatcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer. Such methods include those described in U.S. Pat. No.6,468,798.

Systemic administration of a therapeutic compound as described hereincan also be by transmucosal or transdermal means. For transmucosal ortransdermal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art, and include, for example, for transmucosaladministration, detergents, bile salts, and fusidic acid derivatives.Transmucosal administration can be accomplished through the use of nasalsprays or suppositories. For transdermal administration, the activecompounds are formulated into ointments, salves, gels, or creams asgenerally known in the art.

The pharmaceutical compositions can also be prepared in the form ofsuppositories (e.g., with conventional suppository bases such as cocoabutter and other glycerides) or retention enemas for rectal delivery.

Therapeutic compounds that are or include nucleic acids can beadministered by any method suitable for administration of nucleic acidagents, such as a DNA vaccine. These methods include gene guns, bioinjectors, and skin patches as well as needle-free methods such as themicro-particle DNA vaccine technology disclosed in U.S. Pat. No.6,194,389, and the mammalian transdermal needle-free vaccination withpowder-form vaccine as disclosed in U.S. Pat. No. 6,168,587.Additionally, intranasal delivery is possible, as described in, interalia, Hamajima et al., (1998) Clin. Immunol. Immunopathol., 88(2),205-10. Liposomes (e.g., as described in U.S. Pat. No. 6,472,375) andmicroencapsulation can also be used. Biodegradable targetablemicroparticle delivery systems can also be used (e.g., as described inU.S. Pat. No. 6,471,996).

In one embodiment, the therapeutic compounds are prepared with carriersthat will protect the therapeutic compounds against rapid eliminationfrom the body, such as a controlled release formulation, includingimplants and microencapsulated delivery systems. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid. Such formulations can be prepared using standardtechniques. The materials can also be obtained commercially from AlzaCorporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Kits

Kits according to the present invention are assemblages of separatecomponents. While they can be packaged in a single container, they canbe subpackaged separately. Even a single container can be divided intocompartments. Typically a set of instructions will accompany the kit andprovide instructions for delivering the enzymes, e.g., the GBApolypeptides, intraventricularly. The instructions may be in printedform, in electronic form, as an instructional video or DVD, on a compactdisc, on a floppy disc, on the internet with an address provided in thepackage, or a combination of these means. Other components, such asdiluents, buffers, solvents, tape, screws, and maintenance tools can beprovided in addition to the enzyme, one or more cannulae or catheters,and/or a pump.

Methods of Screening

Also included herein are methods for screening test compounds, e.g.,polypeptides, polynucleotides, inorganic or organic large or smallmolecule test compounds, to identify agents useful in the treatment ofsynucleinopathy disorders that are not associated with a lysosomalstorage disease, e.g., a primary synucleinopathy. In particular, the newscreening assays are designed to locate new compounds that serve asGBA-activating agents for either wild-type or mutant forms of GBA.

As used herein, “small molecules” refers to small organic or inorganicmolecules of molecular weight below about 3,000 Daltons. In general,small molecules useful for the invention have a molecular weight of lessthan 3,000 Daltons (Da). The small molecules can be, e.g., from at leastabout 100 Da to about 3,000 Da (e.g., between about 100 to about 3,000Da, about 100 to about 2500 Da, about 100 to about 2,000 Da, about 100to about 1,750 Da, about 100 to about 1,500 Da, about 100 to about 1,250Da, about 100 to about 1,000 Da, about 100 to about 750 Da, about 100 toabout 500 Da, about 200 to about 1500, about 500 to about 1000, about300 to about 1000 Da, or about 100 to about 250 Da).

The test compounds can be, e.g., natural products or members of acombinatorial chemistry library. A set of diverse molecules should beused to cover a variety of functions such as charge, aromaticity,hydrogen bonding, flexibility, size, length of side chain,hydrophobicity, and rigidity. Combinatorial techniques suitable forsynthesizing small molecules are known in the art, e.g., as exemplifiedby Obrecht and Villalgordo, Solid-Supported Combinatorial and ParallelSynthesis of Small-Molecular-Weight Compound Libraries,Pergamon-Elsevier Science Limited (1998), and include those such as the“split and pool” or “parallel” synthesis techniques, solid-phase andsolution-phase techniques, and encoding techniques (see, for example,Czarnik, (1997) Curr. Opin. Chem. Bio., 1:60-6). In addition, a numberof small molecule libraries are commercially available. A number ofsuitable small molecule test compounds are listed in U.S. Pat. No.6,503,713, incorporated herein by reference in its entirety

Libraries screened using the methods of the present invention cancomprise a variety of types of test compounds. A given library cancomprise a set of structurally related or unrelated test compounds. Insome embodiments, the test compounds are peptide or peptidomimeticmolecules. In some embodiments, the test compounds are nucleic acids.

In some embodiments, the test compounds and libraries thereof can beobtained by systematically altering the structure of a first testcompound, e.g., a first test compound that is structurally similar to aknown natural binding partner of the target polypeptide, or a firstsmall molecule identified as capable of binding the target polypeptide,e.g., using methods known in the art or the methods described herein,and correlating that structure to a resulting biological activity, e.g.,a structure-activity relationship study. As one of skill in the art willappreciate, there are a variety of standard methods for creating such astructure-activity relationship. Thus, in some instances, the work maybe largely empirical, and in others, the three-dimensional structure ofan endogenous polypeptide or portion thereof can be used as a startingpoint for the rational design of a small molecule compound or compounds.For example, in one embodiment, a general library of small molecules isscreened, e.g., using the methods described herein.

In some embodiments, a test compound is applied to a test sample, e.g.,a cell or living tissue or organ, and one or more effects of the testcompound is evaluated. In a cultured or primary cell for example, theability of the test compound to increase levels and/or activity of GBAor PS/SC can be determined. The MES cell-based models described hereincan be used for such screening assays. For example, using MES cellculture plates (96- or 384-well based), small molecule-based chemicallibraries are applied at a test concentration of 1 μM for a period of 36to 48 hours. Cells are lysed and analyzed by sandwich ELISA, e.g., asoutlined in FIGS. 3A to 3D herein to determine the net effect of thesecompounds on the alpha-synuclein protein concentration in each well.

In some embodiments, the test sample is, or is derived from (e.g., asample taken from) an in vivo model of a synucleinopathy disorder asdescribed herein. For example, an animal model, e.g., a rodent modelsuch as a mouse or rat model, can be used. Specifically, the Masliahmouse model of synucleinopathy is suitable (commercially available fromJSW Research in Graz, Austria) (see, Masliah et al., Science, 2000 Feb.18; 287(5456):1265-9.

Methods for evaluating each of these effects are known in the art. Forexample, ability to modulate expression of a protein can be evaluated atthe gene or protein level, e.g., using quantitative PCR or immunoassaymethods. In some embodiments, high throughput methods, e.g., protein orgene chips as are known in the art (see, e.g., Ch. 12, Genomics, inGriffiths et al., Eds. Modern genetic Analysis, 1999, W. H. Freeman andCompany; Ekins and Chu, 1999 Trends in Biotechnology, 17:217-218;MacBeath and Schreiber, 2000 Science, 289(5485):1760-1763; Simpson,Proteins and Proteomics: A Laboratory Manual, Cold Spring HarborLaboratory Press; 2002; Hardiman, Microarrays Methods and Applications:Nuts & Bolts, DNA Press, 2003), can be used to detect an effect on two,three, four, five or more of the polypeptides described herein.

A test compound that has been screened by a method described herein anddetermined to increase levels and/or activity of GBA or PS/SC, or todecrease levels of aS aggregates, can be considered a candidatecompound. A candidate compound that has subsequently been screened,e.g., in an in vivo model of a disorder, e.g., an animal model of asynucleinopathy disorder, and determined to have a desirable effect onthe disorder, e.g., on one or more symptoms of the disorder, can beconsidered a candidate therapeutic agent. Candidate therapeutic agents,once screened in a clinical setting, are therapeutic agents. Candidatecompounds, candidate therapeutic agents, and therapeutic agents can beoptionally optimized and/or derivatized, and formulated withphysiologically acceptable excipients to form pharmaceuticalcompositions.

Thus, test compounds identified as “hits” (e.g., test compounds thatincrease levels and/or activity of GBA or PS/SC) in a first screen canbe selected and systematically altered, e.g., using rational design, tooptimize binding affinity, avidity, specificity, or other parameter.Such optimization can also be screened for using the methods describedherein. Thus, in one embodiment, the invention includes screening afirst library of compounds using a method known in the art and/ordescribed herein, identifying one or more hits in that library,subjecting those hits to systematic structural alteration to create asecond library of compounds structurally related to the hit, andscreening the second library using the methods described herein.

Test compounds identified as hits can be considered candidatetherapeutic compounds, useful in treating synucleinopathy disorders asdescribed herein. A variety of techniques useful for determining thestructures of “hits” can be used in the methods described herein, e.g.,NMR, mass spectrometry, gas chromatography equipped with electroncapture detectors, fluorescence and absorption spectroscopy. Thus, theinvention also includes compounds identified as “hits” by the methodsdescribed herein, and methods for their administration and use in thetreatment, prevention, or delay of development or progression of adisorder described herein.

Test compounds identified as candidate therapeutic compounds can befurther screened by administration to an animal model of asynucleinopathy disorder as described herein. The animal can bemonitored for a change in the disorder, e.g., for an improvement in aparameter of the disorder, e.g., a parameter related to clinicaloutcome.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1 Glycosphingolipids Biochemically Associate with α-Synuclein InVitro

The experiment performed in this example demonstrates the existence ofstable complexes between α-synuclein protein and human braingangliosides, which ultimately become substrates of the GBA enzyme.

Gangliosides are complex glycosphingolipids, which contain aglucocerebroside unit as part of their chemical structure (see, e.g.,Dreisewerd et al., (2005) Anal Chem., 77, 4098-107). As a consequence,glucocerebroside units constitute both a building block and adegradation product in the continuous synthesis-degradation cycle ofgangliosides. Co-incubation of a group of well characterized,brain-derived gangliosides (see, Schlossmacher et al., (2005) N.E.J.M.,352, 728-731, and Dreisewerd et al. (2005)) with recombinant α-synucleinprotein in vitro led to the formation of a complex that was stable underhighly denaturing SDS/PAGE conditions, prompting an electrophoreticshift of the 16 kDa α-synuclein complex to the 19-20 kDa highermolecular weight α-synuclein protein/glucocerebroside complex asindicated by Western blotting (FIG. 1).

FIG. 1 is a representation of a Western Blot, demonstrating the timedependent formation of a stable 19-20 kDa complex (αS/G) between humanbrain gangliosides and α-synuclein protein in vitro. Human brain derivedgangliosides (G) were co-incubated with recombinant human α-synucleinprotein (αS; wild-type) at 4° C. for various periods of time up to 72hours, before being subjected to SDS/PAGE. As a negative control (C),water was incubated with recombinant human α-synuclein protein for 72hours. The time dependent appearance of an upper band migrating at 19-20kDa was noted in the samples incubated with gangliosides, but not withwater, and was interpreted as a stable α-synuclein protein-ganglioside(αS/G) complex. The presence of uncomplexed α-synuclein protein isindicated by a band with the molecular weight of 16 kDa.

These and related findings demonstrated that α-synuclein protein caninteract with glucocerebroside-containing, complex lipids in a mannerthat is highly stable and relatively resistant to the presence of SDS.

Example 2 Glucocerebroside Biochemically Associates with α-SynucleinProtein In Vitro

Human tissue-derived or synthetic Glucocerebroside (aka asglucosylceramide; GC) is co-incubated with recombinant human α-synucleinprotein (αS; wild-type) at 4° C. for various periods of time up to 72hours, before being subjected to SDS/PAGE. As a negative control (C),water is incubated with recombinant human α-synuclein protein for 72hours. The time dependent appearance of an upper band migrating atapproximately 19-22 kDa should be noted in the samples incubated withglucocerebroside. The presence of uncomplexed α-synuclein protein isindicated by a band with the molecular weight of 16 kDa.

These and related findings demonstrate that α-synuclein protein caninteract with glucocerebroside in a manner that is highly stable andrelatively resistant to the presence of SDS.

Example 3 Glucosphingosine Biochemically Associates with α-SynucleinProtein In Vitro

Human tissue—derived or synthetic Glucosphingosine (aka asglucosylsphingosine; GS) is co-incubated with recombinant humanα-synuclein protein (αS; wild-type) at 4° C. for various periods of timeup to 72 hours, before being subjected to SDS/PAGE. As a negativecontrol (C), water is incubated with recombinant human α-synucleinprotein for 72 hours. The time dependent appearance of an upper bandmigrating at approximately 19-22 kDa should be noted in the samplesincubated with glucosphingosine. The presence of uncomplexed α-synucleinprotein is indicated by a band with the molecular weight of 16 kDa.

These and related findings demonstrate that α-synuclein protein caninteract with glucosphingosine in a manner that is highly stable andrelatively resistant to the presence of SDS.

Example 4 Establishment of a Dopamine-Expressing Neural Cell CultureSystem for α-Synuclein Protein Expression

A dopamine-expressing rodent mesencephalic cell culture system (MES23.5cells) was utilized for the establishment of an α-synuclein proteinover-expression system. Previously, these cells had been used by Sharonet al. to create stable cells lines over-expressing αS. However, theseauthors observed that stable αS-transfected MES23.5 cell clonesgradually loose αS expression after passaging for 2-months or more(Sharon R, et al., (2001) PNAS 98, 9110-9115). To avoid this problem, inthis work, MES23.5 cells were transiently transfected each time usingLipofectamine® 2000 (Invitrogen Corp). Since the MES23.5 cells are onlyloosely adherent to tissue culture plastic dishes, the cells werecultured on poly-D-Lysine coated plastic dishes, a measure that was notpreviously used in the literature. Furthermore, Invitrogen Corprecommends transfecting with Lipofectamine 2000 when cells are >80%confluent, it was empirically found here in this study that thetransfection efficiency was much improved by transfecting when cells are50-60% confluent (as measured by transfection efficiency of a GreenFluorescent Protein (GFP)— encoding plasmid in sister wells, visualized24 hours after transfection under a fluorescent microscope).

As shown in FIG. 2A, MES23.5 cells were transiently transfected with afull-length αS encoding SNCA cDNA—plasmid under the control of a CMVpromoter. Cells were transfected with 0, 0.25, 0.5, 1, 5, and 10 μg (per10 cm dish) of plasmid. 24 hours later, cells were washed withTris-buffered saline and lysed in 140 mM NaCl, 50 mM Tris-Hcl, pH 8.0, 1mM EDTA, 0.5% Triton-X100, and 1× protease inhibitors. Lysates werecentrifuged at 100,000×g for 30 min at 4° C.; the top ⅔ of supernatantswere removed and frozen in siliconized tubes at −80° C. Samples were runon SDS/PAGE, using 1 mM DTT as the reducing agent. Expression of the αSprotein was confirmed at 24 hours post-transfection by Western blotting,where cell lysates were probed with a monoclonal antibody against αSprotein (syn-1 antibody, BD Transduction Labs). Expression was shown tobe dependent on the initial amount of plasmid transfected, up to asaturating amount of 5-10 μg per 10 cm dish.

Example 5 Exploratory Studies Using of MES23.5 Cells for theConcomitantly Expression of α-Synuclein and Selected Lysosomal Proteins

The experiment performed in this example (as shown in FIG. 2B)demonstrates that increased levels in cellular GBA protein can reducethe level of neural α-synuclein protein.

MES23.5 cells were transfected with 0.5 μg αS-encoding SNCA cDNA per 10cm dish plus either 1.25, 2.5 or 5 μg (low, medium, or high)GBA-encoding cDNA in the absence or presence of 5 μg ofProsaposin-encoding cDNA. All arms of the experiment were balanced up toa total of 10.5 μg cDNA per 10 cm dish using empty vector cDNA. The GBA-and Prosaposin-encoding cDNA plasmids under a CMV promoter, as well asthe pCMV-XL5 empty vector were purchased from OriGene Technologies, Inc(clones had been fully sequence-verified after isolation and amaxiprep). 24 hours later, cells were lysed and probed for GBA and αSprotein levels. The upper panel in of FIG. 2B3 is a representation of aWestern Blot indicating expression of GBA protein in the absence andpresence of co-transfected prosaposin. GBA was probed using themonoclonal antibody 8E4. GBA over-expression occurred in a slightlygene-dosage dependent way in the absence of prosaposin. In the presenceof prosaposin over-expression, the GBA signal itself was decreased. Thisobservation can be explained by a modification of GBA during itsactivation, leading to its reduced recognizability by the monoclonalantibody employed under these SDS/PAGE/Western blotting conditions, by afaster intra lysosomal degradation rate of GBA after its activation byPS/SC, or it may have occurred as a result of overall reduced cDNAtranscription and translation rates given the concomitant delivery ofthree distinct exogenous cDNA-carrying plasmids.

The lower panel of FIG. 2B shows that GBA, in the absence ofco-transfected prosaposin, lowered the co-expressed α-synuclein proteinlevels at the largest amount of co-transfected GBA cDNA. This observedα-synuclein protein lowering effect by GBA was greatly potentiated bythe co-expression of prosaposin, as indicated by the strong decrease inα-synuclein protein levels at even the lower concentrations (low andmedium) of transfected GBA-encoding cDNA. The bar graph in FIG. 2Cdemonstrates a semi quantitative summary of the data shown in FIG. 2B.

In summary, it was concluded that increased GBA activity under these exvivo cell culture conditions can lower α-synuclein steady-state levels,especially in the presence of elevated PS/SC. Thus, this strategy can beused to lower α-synuclein steady-state levels in vivo, including in thehuman brain that is at risk for—or already affected by—criticallyelevated levels of α-synuclein content, e.g., in a subject having asynucleinopathy disorder. Accordingly, strategies to increase GBAactivity and/or PS/SC levels in vivo represents a novel avenue forneuroprotective treatment of Parkinson's Disease (PD) and relatedsynucleinopathies.

Example 6 Establishment of a First-in-Kind, Sensitive and Precise ELISASystem to Quantitatively Determine α-Synuclein Concentrations inTransfected MES23.5 Cells

For further experiments and investigations, it was desired to decreasereliance on Western blot methods, which are low throughput and havelimited dynamic range, and instead to create a quantitative sandwichELISA (enzyme-linked immune-adsorbent assay) system formedium-throughput quantification of αS with improved sensitivity,optimized specificity and dynamic range.

Sera from 6 rabbits were raised and affinity-purified at OpenBiosystems, Inc. (http://www.openbiosystems.com) against recombinant,full-length human αS. Recombinant αS had been HPLC- and MS-characterizedand subjected to amino acid composition and protein concentrationanalyses. For ELISA, 384-well MaxiSorp plates (Nunc, Inc) were coatedwith 50 μl/well capturing polyclonal Ab (hSA-2) diluted in coatingbuffer (NaHCO3 with 0.2% NaN3, pH 9.6). Following washes with PBS/0.05%Tween-20 (PBS-T), plates were blocked for 2 hours at 37° C. in blockingbuffer (1.125% fish skin gelatin; PBS-T). After 4 washes, samples wereloaded and incubated at 4° C. for 12 hrs. Biotinylated Syn-1 mAb (as theassaying Ab) was generated using 200 μg Sulfo-NHS-LC Biotin (Pierce),diluted in blocking buffer and added to the plate for 2 hrs at 37° C.Following 4 washes, ExtrAvidin phosphatase (Sigma) diluted in blockingbuffer was applied for 1 hr at 37° C. Color development was carried outby using Fast-p-Nitrophenyl Phosphate (Sigma) and monitored kineticallyat OD 405 nm every 5 min for up to 60 min.

Various concentrations of highly purified, recombinant, human αS (r-haS)were used as standards to establish ELISA sensitivity and assay range,as shown in FIG. 3A (r²>0.98).

To optimize a ‘DNA:Lipofectamine® 2000’ ratio with low cell toxicity,MES23.5 cells were transfected with either 0.25, 0.5 or 1 μgαS-encoding, wild-type, human SNCA cDNA plus empty vector cDNA up to atotal of 5.5 μg DNA per 10 cm dish. 24 hours after transfection, celllysates were harvested as described above. For serial dilutions of celllysates, blocking buffer containing 0.5% lysate from vector-transfectedwells was used as diluent, which was also used to create the blank andthe corresponding standard curve of recombinant human αS. Saturationkinetics were examined for identification of time point(s) wherestandards and sample dilutions were in the log phase.

When analyzing these cell lysates by ELISA concentrations in MES-αScells were recorded that showed the expected parallelism after serialdilution, that were SCNA cDNA dose-dependent (both aspects aredemonstrated in the graph of FIG. 3B), and that permitted for the firsttime the precise calculation of the total amount of αS proteinconcentration expressed in living cells (as shown in FIG. 3C).

It was also confirmed that under these refined conditions of cellularexpression the viability of MES23.5 and MES-syn cells was not altered,as measured by LDH in conditioned medium (lactate dehydrogenase, anormally cytosolic enzyme), as a marker of cell leakiness, and bycellular conversion of MTT((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) toformazan, as a marker of intact cellular metabolism. For these standardtoxicity assays, a positive control leading to 100% cell lysis (0.1%Triton-X® 100 treatment) was performed in parallel. The results areshown in FIG. 3D. It was determined that for the range of cDNAconcentrations chosen in all experiments, MES-αS and MES-vector cellswere metabolically fully active in the MTT assay and showed no releaseof cytosol-derived LDH into the conditioned medium of transfected anduntransfected cells. Thus, both assays demonstrated cellular integrity.

Example 7 Synucleinopathy Disease-Related as Well as CatalyticSite-Directed Mutations in GBA Promote the Accumulation of α-Synucleinin Dopaminergic MES Cells

The optimized cell expression/ELISA read-out system described in Example6 was used to examine the effects of over-expression of mutant GBAproteins on αS levels in MES23.5 cells.

MES cells were transfected with 0.5 μg per 10 cm dish of αS-encodingSNCA cDNA-carrying plasmid, plus 5 μg per 10 cm dish of wild-type ormutant, human GBA-encoding plasmid. The GBA variants used were wildtype, N370S, D409H, L444P, E235A, and E340A. These 5 GBA mutants werecreated by site-directed mutagenesis, using the Quickchange® kit(Stratagene), and sequence-verified. The N370S, D409H and L444P areknown to occur (in the homozygous or compound heterozygous state) inGaucher disease in the heterozygous state in Parkinson Disease patientsand/or patients with dementia with Lewy bodies. The E235A and E340Amutant GBA proteins are not known to occur in people. They are directedat the acid/base catalyst and nucleophile, respectively, of the GBAenzyme, and have previously been shown to be catalytically inactive,despite being properly trafficked to the lysosome (Fabrega et al., 2000,Glycobiology, vol 10, pp 1217-1224).

24 hours after transfection, MES cells were lysed as described above andall lysates were analyzed by ELISA. As demonstrated in a composite bargraph that summarize several ELISA experiments (shown in FIG. 4), whencomparing the changes in α-synuclein steady-state to known quantities ofrecombinant α-synuclein protein that was loaded in parallel, it wasrecorded that the co-expression (5 μg/10 cm dish) of wild-type GBA (butnot prosaposin) with αS under these conditions did not significantlychange αS levels (109.7+/−9.88% of vector cDNA control levels). This isin contrast to the result observed in Example 5 above. The observeddiscrepancy can reflect the differences in total DNA transfected in thetwo paradigms (FIG. 2B; FIG. 2C versus FIG. 4), thereby leading tochanges in the DNA:Lipofectamine® 2000 ratio, and in the role ofco-expressed prosaposin (saposin C). It is therefore conceivable thatwild-type GBA can have variable effects on αS levels in these MES23.5cells, depending on the rate of αS import into lysosomes and thecomposition as well as activation state of more than one lysosomalenzyme.

In contrast, the co-transfection with αS of the disease-related N370S,D409H or L444P-carrying mutants of GBA (5 μg per 10 cm dish)consistently led to intracellular α-synuclein accumulation that was121.1+/−4.98%, 269.4+/−56.6%, and 172.7+/−23.02% of control levels(mean+/−standard error of the mean, n=4 (to −6), from 5 independentexperiments), as demonstrated in the bar graph of FIG. 4. These resultshelp explain—for the first time—why people with N370S, D409H or L444Pmutations are more susceptible to sporadic Parkinson's Disease. It isinteresting that the mutation which generally produces the mildest formof Gaucher disease (GD), namely N370S, promoted only a mild accumulationof αS, whereas those associated with a more severe GD phenotype promoteda more prominent accumulation of intracellular αS (see for example, GBAmutant D409H in FIG. 4).

To investigate whether the pro-accumulatory effects of GBA mutations onαS concentrations were due to a trafficking defect causing a moregeneralized cell stress, or a loss of enzymatic function within thelysosome, we next employed two mutants which are properly trafficked tothe lysosome, but exhibit total loss of enzymatic function.Co-transfection with αS of the E235A- and E340A-missensemutation-carrying variants of GBA (5 μg per 10 cm dish) led to theintracellular α-synuclein levels that were 231.0+/−37.14% and156.4+/−19.65% of control vector DNA levels, respectively (mean+/−sem,n=4 (−6), from 5 independent experiments), as demonstrated in the bargraph shown in FIG. 4.

Based on the results of these experiments it appears that activity lossof this non-protease-type lysosomal enzyme contributes at least in partto the αS-accumulatory effect that was induced by human disease-relatedGBA mutants.

Example 8 Expression of Cathepsin D Consistently and SignificantlyReduces α-Synuclein Protein Levels in a Dose-Dependent Manner

The system described in Example 6 was used to examine the effects of aprotease-type lysosomal enzyme, namely cathepsin D, on co-transfected αSlevels.

MES23.5 cells were transfected with 0.5 μg per 10 cm dish of anαS-encoding, wild-type, human SNCA cDNA-carrying plasmid (referred to asMES-hSNCA WT cells in the Western blot shown in FIG. 6), plus either1.25, 2.5 or 5 μg per 10 cm dish of a human Cathepsin D-encoding CTSDcDNA plasmid, which was purchased from OriGene Technologies, Inc., andwas under the control of a CMV promoter. The Cathepsin D clone was fullysequence-verified after isolation and maxiprep. Each transfection armwas balanced with empty vector DNA up to a total of 5.5 μg DNA per 10 cmdish. 24 hours after transfection, cells were lysed, and the resultinglysates were analyzed by the sandwich ELISA described herein.

As demonstrated in FIG. 5, co-expression of human cathepsin D loweredintracellular α-synuclein protein levels. This occurred in a CTSDcDNA-dosage dependent manner, in that increasing amounts ofco-transfected cathepsin D resulted in a progressive lowering ofintracellular α-synuclein levels. When comparing the changes inα-synuclein steady-state to known levels of recombinant α-synucleinprotein that was loaded in parallel, it was calculated that the highestconcentration of cathepsin D over-expression (5 μg/10 cm dish) led to anintra-cellular total α-synuclein level that was 25.3+/−7.0% of controllevels (n=11, from 3 independent experiments). Lower levels of cathepsinD over-expression (1.25 μg/10 cm dish and 2.5 μg/10 cm dish) led tointracellular α-synuclein levels that were 68+/−17.7% and 53+/−16.8% ofcontrol levels, respectively (n=2, from 2 independent experiments).Similarly, human Cathepsin D was able to lower the levels ofco-transfected rat αS, using the same paradigm.

To demonstrate that the αS-lowering effect of Cathepsin D indeedmeasured as high as 75 percent of the total amount of intracellular αSconcentration detectable (and to show that the latter effect was not dueto the chosen ELISA system, the results were confirmed by Westernblotting. As shown in FIG. 6, cell lysates were independently probedwith 2 different anti-synuclein antibodies: the monoclonal syn-1previously described, and a rabbit polyclonal 7071AP (Periquet et al.,(2007) J. Neurosci., 27:3338-46).

Importantly, the co-expression of Cathepsin D with αS for 24 hours didnot lead to the generation of any visible lower or higher molecularweight species, as visualized by syn-1 and 7071AP. The same result wasobtained when using a third antibody, the rabbit polyclonal,affinity-purified hSA-2 (data not shown), and when blots wereover-developed during longer exposure.

To confirm that the effects of Cathepsin D took place in vivo, and notduring the cell lysis procedure, the effects of a potent Cathepsin Dinhibitor, pepstatin A, were examined by its presence in the cell lysisbuffer. A shown in the graph bar of FIG. 5 (first two bars on the left),the inclusion of pepstatin A in the lysis buffer did not change theamount of αS detected in the lysate, thereby demonstrating that theresults described in FIGS. 5 and 6 above were not an artifact of thecell lysis procedure.

To confirm that the effect of Cathepsin D on lowering of αS in MES23.5cells was specific and was not caused by a general decrease in cellularmetabolism and integrity, the MTT and LDH assays were performed onMES-syn cells that had been co-transfected with the highest amount ofCathepsin D-encoding cDNA (5 μg/10 cm dish). Lysis of cells with 0.1%Triton-X® 100 served as a positive control, representing maximal celldeath. MES-syn cells co-transfected with CTSD cDNA exhibited a normalMTT signal that was not different from the control vector transfectedcells (101.3+/−3.91% and 100+/−4.05%, respectively; n=6, from 2independent experiments). Similarly, MES-syn cells co-transfected withCathepsin D exhibited an LDH signal that was identical to that ofcontrol vector transfected cells.

To examine whether Cathepsin D could also reduce the levels of missensemutation-carrying αS proteins, MES23.5 cells were transfected with lowamounts (0.5 μg/10 cm dish) of SNCA cDNA encoding either the A30P, E46Kor A53T variants of α-synuclein which are linked to familial ParkinsonDisease in humans, as well as a S129D and a S129A mutant.Phosphorylation of αS at the Serine 129 residue is known to be apathological hallmark of αS aggregates in vivo (Anderson J et al., 2006,J Biol Chem, vol 281, pp 29739-29752). Mutation of a Ser residue to Aspis known in the art to mimic sustained, serine-based phosphorylation.The S129A mutant, a phosphorylation-incompetent mutant of αS, was alsoincluded for comparison.

As shown in the bar graph of FIG. 7, the co-expression of CathepsinD-encoding CTSD cDNA (5 μg/10 cm dish) with either A30P, E46K, A53T,S129D, or S129A αS caused a similar degree reduction in αS levels forall αS proteins examined, when compared to their co-expression withempty vector DNA. When comparing the changes in α-synuclein steady-stateto well-characterized levels of recombinant α-synuclein protein that wasloaded in parallel, it was estimated that cathepsin D over-expression(at a cDNA concentration of 5 μg/10 cm dish) led to intracellularα-synuclein levels that were 23.98+/−3.57%, 33.08+/−18.51%,39.21+/−14.63%, 34.84+/−11.36% and 34.31+/−13.39% of cognate controllevels, for A30P, E46K, A53T, S129D, or S129A αS polypeptides,respectively (n=2 (−3) from 2-3 independent experiments).

This results suggest (a) that Cathepsin D is capable of also degradingthe mutant forms of αS which occur in familial PD and (b) thatphosphorylation or dephosphorylation at the Ser129 residue of αS doesnot alter the proteolytic (‘synucleinase’) activity exhibited byCathepsin D towards αS.

Of note, residues D98 and Q99 of αS represent the motif by which αS isrecognized by the Lamp2a receptor during chaperone mediated autophagy(CMA); Cuervo et al, 2004, Science, vol 305, pp 1292-1295). Toinvestigate the importance of this motif in the αS-lowering actioninduced by cathepsin D, MES 23.5 cells were also transfected with a cDNA(0.5 μg/10 cm dish) encoding a mutant αS variant, where the D98 and Q99residues had both been changed to Alanine (A) by site-directedmutagenesis (i.e., DQ/AA-variant of αS). When comparing the changes inthe DQ/AA-αS steady-state to well-characterized levels of recombinantα-synuclein protein that were loaded in parallel, it was estimated thatcathepsin D over-expression (5 μg/10 cm dish) led to intracellular DQ/AAαS levels that were 22.14+/−5.32% of the vector control levels, (n=3,from 3 independent experiments; not shown). Based on these results, itappears that either alpha-synuclein also enters the lysosome by a methodother than Lamp2α-mediated CMA, or Cathepsin D exhibits and/or inducesextra-lysosomal activities synucleinase activity.

Example 9 Expression of Cathepsin F Reduces α-Synuclein Protein Levels

The system described in Example 6 was used to examine the effects ofanother lysosomal cathepsin enzyme, namely cathepsin F, onco-transfected αS levels.

MES23.5 cells were transfected with 0.5 μg per 10 cm dish of αS-encodingSCNA cDNA plasmid, plus 5 μg per 10 cm dish of a human CathepsinF-encoding, human CTSF plasmid, which was purchased from OriGeneTechnologies, Inc., and was under the control of a CMV promoter. TheCathepsin F clone was fully sequence-verified after isolation andmaxiprep. 24 hours after transfection, cells were lysed and lysates wereanalyzed by sandwich ELISA.

Twenty-four hours post-transfection, co-expressed human cathepsin Fprotein lowered the intracellular α-synuclein protein concentration, asmeasured by sandwich ELISA. When comparing the changes in α-synucleinsteady-state levels to well-characterized levels of recombinantα-synuclein protein that were loaded in parallel, it was estimated thatcathepsin F over-expression (5 μg/10 cm dish) led to intracellularα-synuclein levels that were 51.7+/−14.1% of control levels (n=3, from 2independent experiments).

To confirm that the effect of Cathepsin F on lowering of αS was specificand was not caused by a general decrease in cellular integrity, the LDHassay was performed on MES-syn cells that had been co-transfected withCathepsin F-encoding CTSF cDNA (5 μg/10 cm dish). Lysis of cells with0.1% Triton-X 100 served as a positive control for cell toxicity,promoting maximal cell death. MES-syn cells co-transfected withCathepsin F exhibited an LDH signal that was less than or equal to thatof control vector transfected cells (data are from 2 independentexperiments; not shown).

Example 10 Increased GBA Activity Prevents Accumulation of α-Synucleinin a Mouse Model

A mouse model in which the wild-type, human α-synuclein protein ismoderately overproduced in the brain can be used as a model for theaccumulation of α-synuclein protein in cell bodies of the brain. GBAactivity level in the central nervous system is increased either bytreating the mice with isofagomine (IFG), an imino sugar that has beenshown to increase GBA activity in mice and humans (Lieberman R et al.,(2007) Nat Chem. Biol. February; 3(2):101-7), or a isofagomine-likesubstance, or by administering or over-expressing GBA protein in themice. The increased GBA activity prevents the age-dependent accumulationof α-synuclein protein in neural cells of the central and/or peripheralnervous system.

Example 11 Increased GBA Activity Provides Therapeutic Effect in aParkinson's Mouse Model

The therapeutic effect of increasing GBA activity in neurons isconfirmed in a novel familial Parkinson's disease model, C3H-Tg(SNCA)83Vle, by showing decreased accumulation of α-synuclein aggregatesin the brain of the test animals. This mouse model expresses mutant A53Thuman α-synuclein under the control of mouse prion (prnp) proteinpromoter. The prnp promoter has been shown to accomplish high levels ofgene expression in most neurons of the central nervous system. By 8months of age, homozygous B6; C3H-Tg(SNCA)83Vle mice begin to developprogressive phenotype and age-dependent intracytoplasmic neuronalinclusions, similar to those seen in patients affected withsynucleinopathies. Increased GBA activity prevents the age-dependentaccumulation of α-synuclein in cell bodies of the brain and reduces thedisease phenotype.

Example 12 Increased Cathepsin D Activity Prevents Accumulation ofα-Synuclein in a Mouse Model

A mouse model in which the wild-type or mutant human α-synuclein proteinis moderately overproduced in the brain can be used as a model for theaccumulation of α-synuclein protein in cell bodies of the human brain.Cathepsin D activity is increased either by treating the micesystemically or by infusion of the brain or stereotactically with asmall molecule activator or stabilizer of Cathepsin D activity, or byadministering or overexpressing Cathepsin D protein or itspre-pro-protein in vivo. The increased Cathepsin D activity prevents theage-dependent accumulation of α-synuclein protein in cell bodies of thebrain. Of course, the same tests can be conducted using other cathepsinpolypeptides, prepolypeptides, and with polynucleotides encoding thesame.

Example 13 Increased Cathepsin D Activity Provides Therapeutic Effect ina Parkinson's Mouse Model

The therapeutic effect of increasing Cathepsin D activity in neurons isconfirmed in a novel familial Parkinson's disease model, C3H-Tg(SNCA)83Vle by showing decreased accumulation of α-synuclein aggregatesin the brain of the test animals. This mouse model expresses mutant A53Thuman α-synuclein under the control of mouse prion (prnp) proteinpromoter. The prnp promoter has been shown to accomplish high levels ofgene expression in most neurons of the central nervous system. By 8months of age, homozygous B6; C3H-Tg(SNCA)83Vle mice begin to developprogressive phenotype and age-dependent intracytoplasmic neuronalinclusions, similar to those seen in patients affected withalpha-synucleinopathies. Increased Cathepsin D activity prevents theage-dependent accumulation of α-synuclein in cell bodies of the brainand reduces the disease phenotype. Of course, this model can be used totest other cathepsins in a similar manner.

Other Embodiments

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A method of treating a subject with asynucleinopathy, but not a clinically diagnosed lysosomal storagedisease, the method comprising administering to a subject anacid-beta-glucocerebrosidase (GBA) polypeptide or a polynucleotideencoding an acid-beta-glucocerebrosidase (GBA) polypeptide in an amounteffective to reduce a level of α-synuclein in the subject's nervoussystem or in the subject's lysosomal compartment.
 2. The method of claim1, wherein the synucleinopathy is a primary synucleinopathy.
 3. Themethod of claim 2, wherein the synucleinopathy comprises any one or moreof: Parkinson's disease (PD); sporadic or heritable dementia with Lewybodies (DLB); pure autonomic failure (PAF) with α-synuclein deposition;multiple system atrophy (MSA); hereditary neurodegeneration with brainiron accumulation; and incidental Lewy body disease of advanced age. 4.The method of claim 1, wherein the synucleinopathy is a secondarysynucleinopathy.
 5. The method of claim 4, wherein the synucleinopathycomprises any one or more of: Alzheimer's disease of the Lewy bodyvariant; Down's syndrome; progressive supranuclear palsy; essentialtremor with Lewy bodies; familial parkinsonism with or without dementia;tau gene and progranulin gene-linked dementia with or withoutparkinsonism; Creutzfeldt Jakob disease; bovine spongiformencephalopathy; secondary Parkinson disease; parkinsonism resulting fromneurotoxin exposure; drug-induced parkinsonism with α-synucleindeposition; sporadic or heritable spinocerebellar ataxia; amyotrophiclateral sclerosis (ALS); and idiopathic rapid eye movement sleepbehavior disorder.
 6. The method of claim 1, further comprisingadministering one or more agents that enhance autophagy of α-synucleincomplexes or enhance degradation of α-synuclein complexes withinlysosomes.
 7. The method of claim 6, wherein the agent comprises an mTORinhibitor.
 8. The method of claim 6, wherein the agent comprisesrapamycin or a rapamycin analog.
 9. The method of claim 6, wherein theagent comprises one or more of everolimus, cyclosporine, FK506, hsc70,N-octyl-4-epi-β-valienamine, and glycerol.
 10. The method of claim 6,wherein the agent comprises a small molecule, a large molecule, apeptide, an antibody, a nucleic acid, or a biologically active fragmentthereof.
 11. The method of claim 1, wherein the subject's nervous systemcomprises the subject's central or peripheral nervous system, or both.